The identification of an organism's total glycosylated protein pool, the glycoproteome, requires selectively and efficiently isolating glycoproteins from biological samples. Various enrichment techniques, such as lectin affinity chromatograpy (Madera et al., (2008) J Sep Sci 31, 2722-2732; Mechref et al., (2008) Methods Mol Biol 424, 373-396), metabolic tagging/click chemistry (Dube et al., (2003) Curr Opin Chem Biol 7, 616-625; Laughlin et al. (2006) Methods Enzymol 415, 230-250), and periodate oxidation/hydrazide chemistry (Zhang et al., (2003) Nat Biotechnol 21, 660-666), have been established for studies in eukaryotes. Among these, chemical coupling by hydrazide chemistry is the most generally applicable. Originally introduced by Zhang and colleagues (Zhang et al., (2003) Nat Biotechnol 21, 660-666), periodate oxidizes the cis-diol of glycans to aldehydes which can then be coupled to a hydrazide resin to form a stable hydrazone bond. The captured glycoprotein can then be analyzed by mass spectrometry. The limitation of this method is the requirement of an enzyme to release enriched protein from functionalized resins. Peptide:N-glycosidase F (PNGase F) has been commonly used for this; however, it cannot cleave the equivalent bonds in most bacterial glycoproteins.
In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This central role makes the PglB protein a hallmark of the likely existence of NLG in an organism and, based on this, genome analysis identified PglB orthologs in numerous bacteria (Nothaft et al. (2010) Microbiology 8:765-778). These include a number of additional bacterial families including some non-ε-proteobacteria. While the presence of PglB is a useful marker, empirical evidence is required to prove NLG exists in a given organism. In certain bacteria, such evidence remains difficult to obtain and full oligosaccharide remains to be determined Free oligosaccharides (fOS) were exploited to demonstrate diverse glycostructures across a variety of Campylobacter and related species (Nothaft et al. (2012) Mol. Cell. Proteomics 11:1203-1219). In the periplasmic space of C. jejuni, free heptasaccharide, structurally identical to that found as an N-linked glycan counterpart, is found that is presumed to be the result of an additional hydrolase activity of PglB (Nothaft et al. (2009) Proc. Natl. Acad. Sci. USA 106:15019-150240). Given these observations, one would hypothesize that identification of fOS from the periplasmic extracts of more distant bacteria would allow prediction of the N-linked glycan structure and provide strong evidence for the broad existence of bacterial NLG systems. This is limited by the need for selective enrichment of fOS from periplasmic extracts and then subsequent structural characterization.
Therefore, there remains a need to identify oligosaccharides from more organisms and in a robust and facile way, using enrichment techniques that do not require the use of a PNGase F.